KR20160096640A - Methods for repairing tissue damage using protease-resistant mutants of stromal cell derived factor-1 - Google Patents

Abstract

The present invention provides compositions comprising a mutant SDF-1 peptide that has been mutated to be resistant to digestion with a substrate cell-derived factor-1 (SDF-1) peptide or protease, but retains chemoattractant activity (e.g., Or a stem cell expressing such a peptide) is used to treat or ameliorate tissue damage.

Description

METHODS FOR REPAIRING TISSUE DAMAGE USING PROTEASE-RESISTANT MUTANTS OF STROMAL CELL DERIVED FACTOR-1 BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

Overall, the invention relates to a method for repairing tissue damage using protease-resistant mutations of SDF-1 or substrate cell-derived factor-1 (SDF-1).

SDF-1 (also known as CXCL12) is a 68 amino acid member of the chemokine family, which is a chemoattractant for resting T-lymphocytes, monocytes and CD34 ⁺ stem cells. SDF-1 is produced in a number of forms: SDF-1? (CXCL12a), SDF-1? (CXCL12b) and SDF-1 ?, which are the results of differential mRNA splicing. The sequences of SDF-1 [alpha] and SDF-1 [beta] are essentially identical, except that SDF-1 [beta] is extended at the C-terminus by four amino acids (Arg-Phe-Lys-Met). The first three exons of SDF-1? Are identical to the exons of SDF-1? And SDF-1 ?. The fourth exon of SDF-l [gamma] is located under the 3200 basepairs from the third exon on the SDF-1 gene locus, and lies between the third exon and the fourth exon of SDF-l [beta]. SDF-1 is initially made with a digested signal peptide (21 amino acids in length) to make the active peptide.

SDF-1 plays an important role in the development of hematopoietic stem cells into the bone marrow during embryonic development and after stem cell transplantation. In addition to its role in stem cell progeny, SDF-1 is also important in cardiac development and angiogenesis. SDF-1-deficient mice die prematurely and have defects in cardiac ventricular septum formation, bone marrow hematopoiesis, and organ-specific angiogenesis. It has also been reported that abnormally low levels of SDF-1 are responsible, at least in part, for wound healing associated with diabetic patients and that the damage can be restored by SDF-I administration at the site of tissue injury.

In a normal adult heart, SDF-1 is expressed constitutively, but expression is down-regulated within a few days after myocardial infarction. Expression of SDF-1 has been shown to increase after 8 weeks of myocardial infarction due to myocardial transplantation of stably transfected cardiomyocytes with overexpression of SDF-1 in combination with G-CSF therapy. This method is associated with more bone marrow stem cells (c-kit or CD34 ⁺ ) and epithelial cells in the heart, resulting in increased vascular density and improved left ventricular function. These studies suggest that the lack of a spontaneous-myocardial repair process may be due to partially inadequate SDF-1 availability. Thus, the transfer of SDF-1 in a controlled manner after myocardial infarction can attract more progenitor cells and thereby facilitate tissue repair.

There is a need in the art for improved methods of promoting wound healing and tissue repair.

SDF-1 is involved in the development of the haematopoietic stem cells and the development of the heart and angiogenesis. In order to stimulate its stem cell mobilization and wound healing effects, the local gradient of SDF-1 is considered to be necessary to promote progenitor cell induction and vascular reformation and repair. We have found that systemic delivery and specifically intravenous ("IV ") delivery of SDF-1 and protease resistant SDF-1 mutations are highly effective in treating tissue damage, It is an amazing result considering. IV delivery has many clinical advantages including, but not limited to, ease of delivery compared to administration of other routes. In addition, we have found that tissue damage (eg, cardiac tissue damage, vascular tissue damage, or wounding) after several minutes of tissue damage events (eg, myocardial infarction) of intravenous administration of SDF-1 or a mutant SDF- , Injury, or tissue damage from disease) is also effective in promoting vascular remodeling and repair, somewhere up to several hours, days, weeks, or months. Here again, our discovery of the efficacy of the composition after a delay period is an unexpected discovery when considering the acute nature of tissue damage in some conditions and diseases.

Thus, although the present invention preserves the ability to act as SDF-1 and its chemically inducible substance, it has been found that proteases, particularly matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP- Mutant SDF-1 peptides mutated in such a way that they are resistant to inactivation by dipeptidyl peptidase IV (DPPIV / CD26), leukocyte elastase, cathepsin G, carboxypeptidase M and carboxypeptidase N Lt; RTI ID = 0.0 > intravenous < / RTI > The methods of the invention may be used, for example, in peripheral vascular disease (PVD, also known as peripheral arterial disease (PAD) or peripheral arterial occlusive disease (PAOD)); Ulcers in the gastrointestinal tract or elsewhere; Wounds caused by accident, surgery, or disease; Chronic heart failure; Tissue damage; Or for the treatment of damaged cardiac tissue as a result of myocardial infarction or other cardiovascular events. The methods of the present invention may also be useful in reducing or treating the potential for tissue damage caused by lesions in wound, ulcer, or diabetic patients. In addition, the methods of the present invention may be used for the treatment of diseases such as regeneration or repair of organs (such as kidney or liver originating from disease or injury), restoration of CNS injuries and inflammatory diseases (e.g. rheumatoid arthritis, Crohn's disease, Host disease). ≪ / RTI >

In one aspect, the chemical formula of the invention the isolated SDF-1 mutant or SDF-1 (mSDF-1) , mSDF-1-Y z, X p -mSDF-1, or X-Y _1-p -mSDF _z (E. G., Tissue damage caused by a disease or condition) in a subject in need thereof by intravenous administration of a composition comprising a mutated form of a SDF-1 peptide having a < RTI ID = 0.0 & Method. SDF-1 is a peptide having an amino acid sequence of at least amino acids 1-8 of SEQ ID NO: 53, which may optionally extend at the C-terminus by all or any portion of the remaining sequence of SEQ ID NO: 53, 53 is an amino acid sequence:

KPX ₃ X ₄ X ₅ X ₆ YRCPCRFFESHVARANVKHLK ILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALNK (SEQ ID NO: 53), wherein X ₃ , X ₄ , X ₅ and X ₆ are any amino acid,

a) X _p is a proteinaceous amino acid (s) or a protease protecting organic group, p is any integer of 1 to 4,

b) Y _z is a proteinaceous amino acid (s) or a protease protecting organic group, z is any integer from 1 to 4,

c) mSDF-1 or mSDF-1-Y _z retains the chemoattractant activity against T cells and inhibits matrix metalloproteinase-2 (MMP-1) activity at a rate of at least 50% 2), matrix metalloproteinase-9 (MMP-9), leukocyte elastase and / or cathepsin G,

d) X _p -mSDF-1 or X _p -mSDF-1-Y _z maintains chemoattractant activity on T cells and degrades dipeptidyl peptides at a rate that is at least 50% less than the rate at which native SDF- Is inactivated by enzyme IV (DPPIV) and is inactivated by MMP-2, MMP-9, leukocyte elastase and / or cathepsin G at a rate that is at least 50% less than the rate of inactivity of native SDF- ,

The isolated mutant form of SDF-1 is administered intravenously in an amount sufficient to treat or ameliorate tissue damage in the subject.

In one particular embodiment, the isolated mutant form of the SDF-1 peptide does not comprise the amino acid sequence of at least amino acids 1-8 of SEQ ID NO: 52.

In one embodiment, X ₃ is valine, histidine, or cysteine. In another embodiment, X < ₄ > is serine or valine. In another embodiment, X < ₅ > is leucine, proline, threonine, or valine. In another embodiment, X < ₆ > is serine, cysteine, or glycine.

In certain embodiments of the methods of the invention, the peptides are X _p -mSDF-1 peptides or X _p -mSDF-1-Y _z peptides wherein X is serine and p is 1. In another embodiment, the peptide is an mSDF-1-Y _z peptide or an X _p -mSDF-1-Y _z peptide wherein Y is serine and z is 1.

In certain embodiments, C-terminal modifications, including the addition of Fc peptides, can be made to any of the SDF-1 peptides described herein, including but not limited to wild-type SDF-1.

In certain embodiments, the mutated form of SDF-1 comprises a sequence as set forth in SEQ ID NO: 63, 67, or 69.

The method of the present invention is characterized in that SDF-1 is a polynucleotide sequence in which A is an isolated mutant form of SDF-1, n is an integer from 0-3 (e.g., 1), L is a linker sequence of 3-9 amino acids, Also characterized is a mutated form of SDF-1, which is a fusion protein having the formula A- (L) _n- Fc, an Fc peptide from the Fc region of an immunoglobulin. In certain embodiments, L is GGGGS (SEQ ID NO: 66). In certain embodiments, the fusion protein can form a biocompatible peptide membrane.

In certain embodiments, the mutated form of SDF-1 is expressed by a stem cell, for example, an adult stem cell, a mesenchymal stem cell, or a mesenchymal precursor cell.

In another embodiment, a composition comprising a stem cell expressing a mutant SDF-1 peptide or an isolated mutant SDF-1 peptide may be administered to a foreign stem cell, for example, an adult stem cell, a mesenchymal stem cell, ≪ / RTI > The exogenous stem cells may be administered prior to, prior to, or subsequent to the administration of the SDF-1-expressing stem cells or the SDF-1 peptide composition.

In certain embodiments of the invention, the disease or condition being treated is selected from the group consisting of cerebral ischemia, traumatic tissue injury, myocardial infarction, peripheral vascular disease, chronic heart failure, diabetes, CNS damage due to injury or disease, (E. G., Rheumatoid arthritis, Crohn ' s disease, or graft versus host disease). Alternatively, the methods of the invention may be used for organ regeneration or repair (e.g., kidney or liver regeneration or repair).

In certain embodiments of the invention, the damaged tissue is cardiac tissue or vascular tissue.

In certain embodiments of the invention, the SDF-1 or mutant SDF-1 protein composition or stem cell composition expressing may be administered to a peripheral vein (e.g., a vein, a leg, a vein in the arm, Or intravenous (IV) or intravenous (IV) intravenous, or intravenous (IV) intravenous, intraperitoneal, intramuscular, intravenous, intraperitoneal, intramuscular,

In certain embodiments of the invention, the SDF-1 or mutant SDF-1 protein composition or stem cell composition expressing is administered within minutes after the initial occurrence of tissue damage or after the onset of a disease or condition, Hours, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours or more, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, Month, three months, six months, one year, no more than two years.

In a further embodiment of the invention, the SDF-1 or mutant SDF-1 protein composition or stem cell composition expressing is administered in combination with a secondary delivery form of SDF-1 or a mutant SDF-1 peptide or stem cell (e.g., Intra-coronary delivery or intra-muscular or intracardiac delivery). Intravenous administration may be second, for example, before or after intraarterial administration. In one embodiment, the SDF-1 or mutant SDF-1 protein composition is first administered intraarterially followed by a period of time ranging from several minutes to one hour to several hours, up to one day, up to one week, up to one month, After a period of time, the SDF-1 or mutant SDF-1 protein composition is administered intravenously. Intraarterial administration may be repeated prior to intravenous administration or for a period of time following intravenous administration.

A SDF-1 or mutant SDF-1 protein composition or stem cell composition expressing may be administered more than once to ameliorate one or more symptoms of the disease or condition. The SDF-1 or mutant SDF-1 composition or stem cell composition expressing may be administered one or more times until the tissue damage is reduced, tissue is restored, or new blood vessel formation occurs.

In various embodiments, the disease or condition is traumatic tissue damage, myocardial infarction, peripheral vascular disease. In a further embodiment, the disease or condition is cardiovascular disease.

In certain embodiments of the invention, the damaged tissue is cardiac tissue or vascular tissue.

By "sufficient amount" is meant the amount of therapeutic agent (eg, mSDF-1 peptide), alone or in combination with other therapies, needed to treat or ameliorate a disorder or condition in a clinically relevant manner. In one embodiment, a sufficient amount of SDF-1 or a mutant SDF-1 peptide of the invention is an amount that promotes wound healing or tissue repair or new blood vessel formation (e.g., angiogenesis). For example, the sufficient amount of the therapeutic agent used to practice the present invention for the treatment of tissue damage depends on the manner of administration, the age of the subject, and the overall health condition. In the end, the medical professional prescribing these treatments will determine the appropriate amount and dosage regimen.

Means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the entire length of the nucleic acid or polypeptide Quot; means a portion of a nucleic acid or polypeptide. The nucleic acid fragment may contain up to the full length of the nucleic acid, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 nucleotides. The polypeptide fragment may contain up to the full length of, for example, 10, 20, 30, 40, 50, or more than 60 amino acids, polypeptides. Fragments may be modified as described herein and as known in the art.

Intravenous administration, "" IV administration, "or" IV treatment "refers to administration of a substance into the vein Intravenous administration may involve direct injection into the vein via a needle connected to a direct syringe or connected with a strand of vessel and a vessel (e.g., a sterile vessel housing the pharmaceutical composition to be administered).

"Intraarterial administration" refers to the administration of a substance into an artery (eg, coronary artery (eg, intracoronary administration)). Intraarterial administration may include intraarterial injection or infusion, or administration via an intraarterial catheter.

"Intramuscular administration " means administration of a substance to the muscle.

"Intra myocardial administration" means administration of a substance to the myocardial or cardiac muscle.

"Pharmaceutically acceptable carrier" means a physiologically acceptable carrier for the subject to be treated, while maintaining the therapeutic properties of the co-administered compositions. One exemplary pharmaceutically acceptable carrier material is saline. Other physiologically acceptable carriers and combinations thereof are known to those skilled in the art and are described, for example, in Remington ' s Pharmaceutical Sciences (20 ^th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA) do.

"Promoting wound healing" or "promoting tissue repair" means increasing, improving, increasing, or inducing wound, healing, or repair of a wound or damaged tissue. Wound or tissue damage can be the result of any disorder or condition (e.g., disease, injury, or surgery) and can be found at any location in the subject (e.g., intracranial or traumatic). For example, wound or tissue damage may be the result of, for example, cardiovascular conditions such as myocardial infarction, and damaged tissue may be cardiac tissue. Alternatively, wound or tissue damage may be the result of peripheral vascular disease or diabetes.

"Protein," "polypeptide," "polypeptide fragment," or "peptide" refers to a protein or polypeptide that comprises all or part of a naturally occurring polypeptide or peptide, Quot; means any chain of two or more amino acid residues that constitutes a generative polypeptide or peptide. Polypeptides or peptides may be referred to as " isolated "or" substantially pure "when a physical, mechanical, or chemical method is used to remove the polypeptide from the cellular component. &Quot; isolated polypeptide, "" substantially pure polypeptide," or "substantially pure and isolated polypeptide" means that it is removed from the cell component in the absence of more than 60 wt% protein and naturally- Is typically considered pure. The polypeptide may be pure at 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, or 99 wt% or more. Substantially pure polypeptides can be obtained by standard techniques, for example, by extraction from a natural source (such as a cell line or biological fluid), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the polypeptide Can be. Purity can be measured by any suitable method, for example, by column chromatography, polyacrylamide gel electrophoresis, or high pressure liquid chromatography (HPLC) analysis. Alternatively, the polypeptide is considered to have been altered by human intervention placed in its non-natural site, or isolated if introduced into more than one cell.

As defined above, the peptides and polypeptides of the present invention include all "mimetic" and "peptide mimetic" forms. The terms "mimetic" and "peptide mimetic" refer to synthetic chemical compounds having substantially the same structural and / or functional properties of the peptides or polypeptides of the invention. The mimetics can be composed entirely of synthetic, non-natural analogs of amino acids, or non-natural analogs of chimeric molecules and amino acids of natural amino acids. The mimetics may incorporate any amount of conservative substitution, as long as such substitution does not substantially alter the structure or activity of the mimetics.

By "prevention" or "reduction of likelihood" is meant reducing the severity, frequency and / or duration of a disease or disorder (eg, myocardial infarction or peripheral vascular disease) or symptoms thereof.

The term "protease protective organic group" refers to the chemoattractant activity of unmodified SDF-1 when added to the N-terminal amino acid of the mutated form (mSDF-1) of SDF-1 or SDF- For example, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% or more (for example, a measurement of Jurkat T cell migration 45, 40, 35, 30, 25, 20, 20, 30, 40, 50, 50, 50, 60, or 80 percent of the inactive rate of unmodified SDF- Refers to an organic group that results in a modified peptide that is inactivated by an enzyme (e.g., DPPIV) at a rate of 15, 10, 5, or less than 1%.

"Protease-resistant" is intended to include one or more modifications in the primary sequence of its amino acid compared to native or wild-type peptides or polypeptides (e.g., native or wild-type SDF-1) Or a peptide or polypeptide that exhibits increased resistance to proteolysis as compared to a polypeptide. "Increased protease-resistant" means an increase measured in vitro or in vivo, as compared to a peptide or polypeptide without amino acid sequence alteration. Increased resistance to proteases can be measured using specific proteases (e. G., ≪ RTI ID = 0.0 > e. ≪ / RTI > For example, by testing for activity or expression at a subsequent exposure to MMP-2, MMP-9, DPPIV, leukocyte elastase, cathepsin G, carboxypeptide degradase M, or carboxypeptide degradase N . Typically, the increase in protease resistance is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 5% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more.

By "proteinaceous" is meant that the amino acid of the polypeptide or peptide is selected from the group consisting of alanine (A), arginine (R), asparagine (N), aspartate (D), cysteine (C), glutamic acid (E), glutamine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine W), tyrosine (Y), or valine (V).

The term " SDF "or" SDF-1 "refers to a sequence of SEQ ID NO: 52 or any of a plurality of SDF forms (for example, SDF- ) And SDF-y). ≪ / RTI > SDF-1 [beta] contains an additional four amino acid residues, Arg-Phe-Lys-Met at the C-terminus of SDF-l [alpha]. The first three exons of SDF-1? Are identical to the exons of SDF-1? And SDF-1 ?. The fourth exon of SDF-l [gamma] is located under the 3200 basepairs from the third exon on the SDF-1 gene locus, and lies between the third exon and the fourth exon of SDF-l [beta]. Although SEQ ID NO: 52 shows the sequence of SDF-la, this sequence may be extended at the C-terminus to include additional amino acid residues. The present invention includes mutations of SDF-l [alpha], SDF-l [beta] and SDF-y. For purposes of the present invention, the term "SDF" or "SDF-1" refers to the active form of the peptide, i.e., after degradation of the signal peptide.

(where N is a letter designation of the original amino acid, q is the position of the peptide from the N-terminus of the peptide, and N 'is the substitution of N for mSDF-1, mSDF, or SDF One amino acid) refers to a mutant SDF-1 peptide. The peptide mutated by the addition of an amino acid (e.g., one or more amino acids) at the N-terminus is referred to as "X _p -R ", where X is a proteinaceous amino acid or a protease protecting organic group, p is an integer, (For example, SDF-1 or mSDF-1). For example, "S-SDF-1" or "S-mSDF-1" are SDF-1 or mSDF-1 molecules with a serine residue added at the N-terminus, respectively. The peptide mutated by the addition of an amino acid (e.g., one or more amino acids) at the C-terminus is referred to as "RY _{z &} quot ;, where Y is a proteinaceous amino acid or protease protective organic group, z is an integer, (e.g., SDF-1, mSDF-1 , or X -mSDF _p-1) is referred to as Im). Unless otherwise indicated, all pharmaceutically acceptable forms of the peptides can be used, including any pharmaceutically acceptable salts.

By "SDF-1 or mutant SDF-1 peptide of the invention" is meant any wild-type SDF-1 (including isoform) or mutant SDF-1 peptide described herein. The term also includes compositions (e. G., Pharmaceutical compositions) comprising the wild-type SDF-I or mutant SDF-I peptides described herein.

"Stem cell" refers to an undifferentiated biological cell that is pluripotent and can be further divided to produce more stem cells, which can differentiate into various differentiated cells. The term includes embryonic stem cells, adult stem cells, mesenchymal stem cells and mesenchymal precursor cells. "Mesenchymal stem cells" means pluripotent stromal cells; "Mesenchymal precursor cells" are STRO-1 ("STRO-1 +"), a mesenchymal precursor cell characterized by the presence of cell surface markers.

"Subject" means a mammal, including, but not limited to, a human or non-human mammal such as cattle, horses, dogs, sheep, cats.

By "sustained release" or "controlled release" is meant that the therapeutically active component is administered at a therapeutically beneficial level (but less than a toxic level) of the component, such as from about 12 hours to about 4 weeks (eg, 12 hours, 24 hours, 48 hours, 1 week, 2 weeks, 3 weeks, or 4 weeks), thus providing a controlled rate to provide, for example, a 12-hour to 4-week dosage form ≪ / RTI >

"Treatment" or "improvement" means administering the pharmaceutical composition for therapeutic purposes or administering the treatment to a subject already suffering from a disorder to improve the condition of the subject. "Treating a disorder" or "ameliorating a disorder" means that a symptom associated with a disorder and disorder is, for example, alleviated, reduced, cured, or placed in a state of driveability. The degree of such improvement or treatment, as compared to an untreated equivalent control, may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% , 90%, 95%, 99%, or 100%.

Other features and advantages of the invention will be apparent from the detailed description and from the claims.

Figure 1 is a bar graph showing that SSDF-1 (S4V) was delivered intravenously and that 7 days after ischemic reperfusion injury improved ejection fraction (EF) by 10 percentage points compared to PBS control.
FIG. 2 shows the results of intravenous administration of SSDF-1 (S4V) immediately after infarction following intravenous administration of SSDF-1 (S4V) 4 weeks after infarction in a micro Yucatan pig model of ischemic reperfusion injury Was improved by 2.7 percentage points at 12 weeks after infarction compared to the PBS control group. A 1-sided T-test was performed; p <0.05; n = 5 pigs / county.

Detailed description

The present invention is based on the discovery that the repair of damaged tissue, e. G., Damaged cardiac tissue, is inhibited by wild type SDF-I or enzymatic degradation (e.g., one or more MMP-2, MMP-9, DPPIV, leukocyte elastase, cathepsin G, Is promoted by intravenous administration of the mutated SDF-1 to increase the resistance to < RTI ID = 0.0 > IL-1 < / RTI > degradation by carboxypeptide degrading enzyme M or carboxypeptide degrading enzyme N. Such peptides may be administered as isolated peptides, with or without a pharmaceutically acceptable carrier. In addition, we have surprisingly found that after an initial incidence of tissue injury or the onset of a disease or condition, or an initial occurrence of tissue damage from within minutes after recognition, or diagnosis, or the onset of a disease or condition, , 3 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours or more, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 1 month, 2 months, 3 months , Six months, one year, and more than two years were also useful in promoting recovery of damaged tissue. This approach can be used to treat damaged tissue resulting from any type of injury or disease.

Intravenous  administration

The SDF-1 or mutant SDF-1 peptide-containing compositions or stem cell compositions expressing used in the methods of the present invention can be administered by intravenous (IV) injection or by implantable devices (e.g., catheters) ≪ / RTI > Intravenous administration generally refers to administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, in the form of a pharmaceutical composition, usually in the form of a peripheral vein (e.g., a vein in the arm, a leg, a vein in the back of a hand, Including, but not limited to, intravenous, intraperitoneal, intrathecal, intravenous, intraperitoneal, intrathecal, intrathecal, Intravenous administration may also include administration by an inserted central catheter, a central venous catheter, or an implantable port.

Peripheral IV tubing can be placed in the distal vein (e.g., in the chest or abdomen) through the skin using a cannula-over-needle device, for example, in which a flexible plastic cannula is mounted on a metal trocar And a short catheter (several centimeters long) inserted into the vein. The portion of the catheter that remains outside the skin is called the connecting hub; It may be connected to a syringe or an intravenous infusion tube. The potted cannula has a scanning port at the top that can be used to administer the SDF-1 mutant SDF-1 peptide of the present invention.

Peripheral injected central catheter (PICC) tubing is used when an IV approach is needed over an extended period of time or when the material to be injected causes premature damage and premature loss of peripheral IV and to attempt a conventional central tube Used when it is too dangerous.

The IV delivery method of the present invention also includes a central venous catheter, for example, wherein the catheter is inserted into the subclavian jugular vein or femoral vein and proceeds toward the heart until it reaches the superior vein or right atrium.

Another central IV delivery method is through the use of a central IV tube that flows through the catheter into the large vein, usually the superior or inferior vein, or the tip of the heart in the right atrium.

Another type of central tube useful in the IV delivery method of the present invention is the Hickman line or Broviac line which is "tunneled " undercut to be inserted into the destination vein and subsequently appearing a short distance apart, It is a catheter.

Portable ports are also used in the IV delivery of SDF-1 and mutant SDF-1 peptide compounds or stem cells of the invention. Ports that can be implanted are central lines without external connectors; Instead, it has a small reservoir covered with silicone rubber and is implanted subcutaneously. The peptide compound is administered intermittently by placing small needles through the skin and piercing the silicone with a reservoir. Ports can remain in the subject's body for weeks, months, or even years. Intermittent infusion is another type of intravenous administration that can be used when the subject only needs administration of the SDF-1 and mSDF-1 peptide compounds or stem cells of the present invention at a given time.

SDF-1 or mSDF-1 peptide-containing compositions or stem cell compositions expressing can be administered in one vein or in several veins. The SDF-I or mSDF-I peptide-containing composition or stem cell composition expressing may be administered to a subject in need thereof, for example, for at least 1 minute, 1 to 5 minutes, 10 to 20 minutes, 20 to 30 minutes, RTI ID = 0.0 > intravenous < / RTI > Administration can be intermittently repeated to achieve or maintain the expected benefit. Repeated dosing times are based on the response of the subject, for example, by monitoring the symptoms associated with tissue damage. A therapeutically effective dose or amount of the SDF-1 or mSDF-1 peptide-containing composition or stem cell composition to be provided may be divided into two or more doses and the dose may be divided into two or more Can be administered intravenously.

SDF -1 and protease-resistant mutations

Formally known as chemokine (C-X-C motif) ligand 12 (CXCL12), SDF-1 is a small cytokine belonging to the chemokine family. SDF-1 is produced in multiple forms of SDF-1? (CXCL12a), SDF-1? (CXCL12b) and SDF-1? By alternating splicing of the same gene.

Unmutated SDF-1 alpha has the following sequence:

(SEQ ID NO: 52) K P V S L S Y R C P C R F F E S H V A R N V K H L K I L N T P N C A L I V R L K N N R Q V C I D P K L K W I Q L Y K L K K (SEQ ID NO: 52)

The SDF-1 peptides described herein can be produced by a method comprising the steps of: contacting a peptide with, for example, a matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), dipeptidyl peptidase IV (DPPIV) 1 < / RTI > peptide with a mutation that allows it to become resistant to leukocyte elastase, cathepsin G, carboxypeptide degrading enzyme M, or carboxypeptide degrading enzyme N. In the methods of the invention, the unmutated SDF-1 may be administered by intravenous delivery for the treatment or amelioration of tissue damage.

The method of the invention is characterized by the mutation form of SDF-1 (mSDF-1), which is characterized by a change in the third, fourth, fifth and / or sixth amino acid residues from the N-terminus of unmutated SDF- . The mSDF-1 peptide of the present invention has at least amino acids 1-8 of SEQ ID NO: 53 and may be extended at the C-terminus by all or any portion of the remaining sequence of SEQ ID NO: 53 having the following sequence:

KPX ₃ X ₄ X ₅ X ₆ YRCPCRFFESHVARANVKHLK ILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALNK (SEQ ID NO: 53) wherein X ₃ , X ₄ , X ₅ and X ₆ are any amino acid residues.

In certain embodiments, X ₃ is valine, histidine, or cysteine.

In certain embodiments, X ₄ is serine or valine.

In certain embodiments, X < ₅ > is leucine, proline, threonine, or valine.

In certain embodiments, X ₆ is serine, cysteine, or glycine.

For example, the mSDF-1 peptide may comprise a mutation at a fourth (e.g., Ser to Val) and / or a fifth (e.g., Leu to Pro) amino acid position.

KPV V LSYRCPCRFFESHVARANVKH LKILNTPNCALQIVARLKNNN RQVCIDPKLKWIQEYLEKALN K (SEQ ID NO: 63)

KPVS P SYRCPCRFFESHVARANVKHL KILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALNK (SEQ ID NO: 64)

KPV V P SYRCPCRFFESHVARANVKHL KILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALNK (SEQ ID NO: 65)

In another embodiment, the mSDF-1 peptide may comprise a Val → His (SEQ ID NO: 54) or Val → Cys (SEQ ID NO: 55) mutation at the third amino acid position.

KP H SLSYRCPCRFFESHVARANVK HLKILNTPNCALQIVARLKNN NRQVCIDPKLKWIQEYLEKAL NK (SEQ ID NO: 54)

KP C SLSYRCPCRFFESHVARANVK HLKILNTPNCALQIVARLKNN NRQVCIDPKLKWIQEYLEKAL NK (SEQ ID NO: 55)

In another embodiment, the mSDF-1 peptide may comprise a Leu → Thr (SEQ ID NO: 56) or Leu → Val (SEQ ID NO: 60) mutation at the fifth amino acid position.

KPVS T SYRCPCRFFESHVARANVKHL KILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALNK (SEQ ID NO: 56)

KPVS V SYRCPCRFFESHVARANVKHL KILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALNK (SEQ ID NO: 60)

In another embodiment, the mSDF-1 peptide may comprise a Ser to Cys (SEQ ID NO: 61) or Ser to Gly (SEQ ID NO: 62) mutation at the sixth amino acid position.

KPVSL C YRCPCRFFESHVARANVKHLK ILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALNK (SEQ ID NO: 61)

KPVSL G YRCPCRFFESHVARANVKHLK ILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALNK (SEQ ID NO: 62)

The methods of the invention may also include peptides encompassing any combination of mutations described herein. For example, the mSDF-1 peptide may comprise a Val → Cys mutation at the third amino acid position of SEQ ID NO: 53 and a Ser → Cys mutation at the sixth amino acid position of SEQ ID NO: 53.

Mutations made in the SDF-1 peptide to impart protease resistance, for example, by creating a -mSDF X _p-1, for example, the N- terminal moiety of mSDF-1 peptide (a) (e.g., A proteinaceous amino acid or a protease protective organic group). For example, when X is R ¹ - (CH ₂ ) _d - (d is an integer of 0 to 3, R ¹ is hydrogen (when R ¹ is hydrogen, d has to be at least 1) Or branched C ₁ -C ₃ alkyl, straight or branched C ₂ -C ₃ alkenyl, halogen, CF ₃ , -CONR ⁵ R ^4, -COOR ^5, -COR ^5, - (CH ₂ ) _q NR ⁵ R ^{_{4; - (CH 2) q}} SOR 5; - (CH 2) q SO 2 R 5, - (CH 2) q SO 2 NR 5 R 4 and OR ⁵ days and may, in which, R ⁴ and R ⁵ are each independently hydrogen or linear or branched C ₁ -C ₃ alkyl group. the organic group in the case used for the X, p must be 1. X is proteinaceous may represent an amino acid, e.g., 1-10 Amino acid (s) are selected from the group consisting of SDF-1 (e. G., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, For example, mSDF-1, and one or more of these added amino acids may be substituted with a protease protective organic group. For example, a proteinaceous amino acid (e.g., three Lean) or protease protective organic group can be used to inhibit SDF-1 (e.g., < RTI ID = 0.0 > For example, mSDF-1). The following sequence represents an exemplary SDF-1 mutation with a serine amino acid added at the N-terminus.

S KPX ₃ X ₄ X ₅ X ₆ YRCPCRFFESHVARANVKHLK ILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALNK (SEQ ID NO: 68) wherein X ₃ , X ₄ , X ₅ and X ₆ are any amino acid residues.

In certain embodiments, X ₃ is valine, histidine, or cysteine.

In certain embodiments, X ₄ is serine or valine.

In certain embodiments, X < ₅ > is leucine, proline, threonine, or valine.

In certain embodiments, X ₆ is serine, cysteine, or glycine.

Specific examples of the sequence include:

S KPV V LSYRCPCRFFESHVARANVKH LKILNTPNCALQIVARLKNNN RQVCIDPKLKWIQEYLEKALN K (SEQ ID NO: 69)

S KPVS P SYRCPCRFFESHVARANVKHL KILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALNK (SEQ ID NO: 70)

S KPV V P SYRCPCRFFESHVARANVKHL KILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALNK (SEQ ID NO: 71)

S KP H SLSYRCPCRFFESHVARANVK HLKILNTPNCALQIVARLKNN NRQVCIDPKLKWIQEYLEKAL NK (SEQ ID NO: 72)

S KP C SLSYRCPCRFFESHVARANVK HLKILNTPNCALQIVARLKNN NRQVCIDPKLKWIQEYLEKAL NK (SEQ ID NO: 73)

S KPVS T SYRCPCRFFESHVARANVKHL KILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALNK (SEQ ID NO: 74)

S KPVS V SYRCPCRFFESHVARANVKHL KILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALNK (SEQ ID NO: 75)

S KPVSL C YRCPCRFFESHVARANVKHLK ILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALNK (SEQ ID NO: 76)

S KPVSL G YRCPCRFFESHVARANVKHLK ILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALNK (SEQ ID NO: 77)

Mutations made to the SDF-1 peptide to confer protease resistance can be detected, for example, by generating mSDF-1-Y _z or X _p -mSDF-1-Y _z , -Terminal (e. G., A proteinaceous amino acid) at the < / RTI > end. Y can represent a proteinaceous amino acid and can be, for example, 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1) amino acid (s) is a SDF-1 (e. g., is added to the C- terminus of mSDF-1 or X -mSDF _p-1). For example, a proteinaceous amino acid (e. G., Serine) may be conjugated to, for example, a carboxypeptidase M (e.g., serine) without substantially altering chemoattractant activity or resistance to another protease or carboxy peptide N-decomposing enzyme can be added to the C- terminus of the SDF-1, mSDF-1, or X _p -mSDF-1 to confer resistance to degradation. In one embodiment, the invention features an isolated mSDF-1-Y _z or X _p -mSDF-1-Y _z peptide, wherein SDF-1 comprises the amino acid sequence of SEQ ID NO: 53. However, C-terminal modifications can be made to any of the SDF-1 peptides and SDF-1 described herein. The mutated SDF-1 peptides described herein retain their ability to act as chemoattractants but are resistant to enzymatic (e.g., protein hydrolysis) digestion. The mSDF-1 peptide may be at least 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% sensitive (Determined by, for example, the effective concentration required to obtain, for example, 50% of the maximal response in a test for Jurkat T cells or any other chemotaxis measurement method known in the art) . Loss of chemoattractant activity may be due to, for example, degradation of MMP-2, MMP-9, leukocyte elastase, DPPIV, cathepsin G, carboxypeptide degrading enzyme M, or carboxypeptide degrading enzyme N. The rate of inactivation of mSDF-1 may be, for example, less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1% of the rate of inactivation of SDF-1.

The mutated SDF-1 peptide may be resistant to degradation by, for example, MMP-2, MMP-9, DPPIV, leukocyte elastase, cathepsin G, carboxypeptide degrading enzyme M, or carboxypeptide degrading enzyme N have. Thus, it is ideally suited for use at sites such as, for example, damaged tissue (e. G., Damaged cardiac tissue), and proteolytic enzymes are present at high concentrations or delivered to the site via blood or plasma. Therefore, mutated SDF-1 peptides are suitable for intravenous administration because of the improved stability of such peptides.

The protease-resistant SDF-1 peptides described herein may contain amino acid or natural modifications, such as by post-translational modification, or by chemical modification using techniques known in the art have. Modifications can occur anywhere in the polypeptide, including polypeptide backbones, amino acid side chains, and amino- or carboxy-termini. The same type of modification may be present at the same or varying degrees in several parts of a given polypeptide, and the polypeptide may comprise more than one type of modification. Modifications include, for example, PEGylation, acetylation, acylation, addition of acetomidomethyl (Acm) groups, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, , Covalent attachment to a fibrin, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a drug, covalent attachment of a label (fluorescent or radioactive label), attachment of a lipid or lipid derivative, phosphatidyl The formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, formation of GPI anchor, formation of covalent crosslinks, formation of covalent bonds, But are not limited to, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, -RNA mediated transfer of the amino acid to the protein is added (e. G., Aralkyl groups biotinylated) a and ubiquitination. The post-translational modifications also include the addition of a polymer to stabilize the peptide or to improve pharmacokinetics or pharmacodynamics. Exemplary polymers include, for example, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co- Poly (ethylene glycol), polylactide (PLA), poly (lactide-co-glycolide), poly (ethylene glycol) Glycolide) (PLGA), polyglutamic acid (PGA), and polyorthoesters.

Fusion protein

The method of the invention the SDF-1, mSDF-1, X p -mSDF-1, with a random peptide sequence of mSDF-1-Y _{z, p} -mSDF or X-Y _1-z is IgG (as disclosed herein For example, a fusion protein linked to the Fc region of human IgGl) can also be used. Alternatively, the Fc region may be derived from other animal or human IgA, IgM, IgE, or IgD, including pigs, mice, rabbits, hamsters, goats, rats and guinea pigs. The Fc region of IgG includes the CH2 and CH3 domains of the IgG heavy chain and hinge region. The hinge acts as a flexible spacer between two parts of the Fc fusion protein, allowing each part of the molecule to function independently. The Fc region used in the present invention can be produced, for example, in the form of a monomer or a dimer.

An exemplary Fc fusion peptide is S-SDF-1 (S4V) -Fc having the amino acid sequence: The GGGGS linker (SEQ ID NO: 66) appears bold and the Fc peptide is underlined.

SKPVVLSYRCPCRFFESHVAR ANVKHLKILNTPNCALQIVAR LKNNNRQVCIDPKLKWIQEYL EKALNK GGGGS VDKTHTCPPCPAPELLGGPSV FLFPPKPKDTL Met ISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQG NVFSCSV HEALHNHYTQKSLSLSPGK Met (SEQ ID NO: 67)

Other non-limiting examples of Fc fusion peptides include, but are not limited to, SDF-1 (S4V) -Fc, SDF-1 (L5P) -Fc, SDF- , SDF-I-Fc, S-SDF-I-Fc, and SDF-I-Fc.

All such proteins are included in the term " SDF-1 and mSDF-1 proteins of the invention "or" peptides of the invention ".

Peptide synthesis

SDF-1 or protease-resistant mutant SDF-1 peptides used in the methods of the present invention can be prepared by using the chemical properties of, for example, the standard N-tert-butyoxycarbonyl (t-Boc) Lt; RTI ID = 0.0 > n-methylpyrrolidone < / RTI > Exemplary methods for synthesizing peptides are described, for example, in U.S. Patent 4,192,798, which is incorporated herein by reference; 4,507,230; 4,749,742; 4,879,371; 4,965,343; 5,175,254; 5,373,053; 5,763,284; And 5,849,954. Such peptides can also be made using recombinant DNA technology.

Once the peptide is synthesized, it can be purified using methods such as, for example, HPLC on a reversed phase column. Purity can also be measured by HPLC, and the presence of the correct composition can be determined by amino acid analysis. Suitable purification methods for mSDF-1 peptides are described, for example, in U.S. Patent Application Publication No. 2008/0095758, which is incorporated herein by reference.

Fusion proteins can be chemically synthesized or made using recombinant DNA technology. Other non-limiting examples of Fc fusion peptides include, but are not limited to, SDF-1 (S4V) -Fc, SDF-1 (L5P) -Fc, SDF- , SDF-I-Fc, S-SDF-I-Fc, and SDF-I-Fc.

SDF -1 peptide < / RTI > Stem Cells

The present invention relates to a method for the production of stem cells and / or their genetically modified progeny cells (e. G., Cells) to express and / or secrete the peptides of the invention (e. G., SDF-1 or protease-resistant mutant SDF- Lt; / RTI > Any suitable stem cells can be genetically modified to express and / or secrete the peptides of the invention, including, for example, adult stem cells, mesenchymal precursor cells (MPCs) and mesenchymal stem cells (MSCs) . In some embodiments, the stem cells can naturally express the basal level of wild-type SDF-1 and the genetic modification can be carried out such that the stem cells express an increased level of wild-type SDF-1 and / or the protease-resistant mutant SDF1- To express the peptide.

Methods for genetically transforming cells, e. G., Stem cells, will be apparent to those skilled in the art. For example, a nucleic acid to be expressed in a cell is operatively linked to a promoter that induces expression in the cell. For example, the nucleic acid is linked to a promoter operable in various cells of a subject, such as, for example, a viral promoter, for example, a CMV promoter (e.g., CMV-IE promoter) or SV-40 promoter. In other cases, the promoter may be specifically operable in certain types of stem cells. Additional suitable promoters will be known in the art and applied as needed to this embodiment of the disclosure.

In one embodiment, the nucleic acid is provided in the form of an expression construct. As used herein, the term "expression construct" refers to a nucleic acid having the ability to confer expression on a nucleic acid operatively linked in a cell (e.g., a reporter gene and / or a counter-selectable reporter gene) Quot; In the context of this disclosure, expression constructs include plasmids, bacteriophages, phagemids, cosmids, viral sub-genomes or genomic fragments, or other nucleic acids capable of maintaining and / or replicating heterologous DNA in a format that can be expressed It can be understood that

Methods for the construction of expression constructs suitable for carrying out the present disclosure will be apparent to those skilled in the art and are described, for example, in Ausubel et al., Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987] or Sambrook et al. [Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001]. For example, each component of the expression construct can be amplified from a suitable template nucleic acid, for example, using polymerase chain reaction (PCR), and subsequently amplified from a suitable expression construct, such as, for example, a plasmid or phagemid Lt; / RTI >

Vectors suitable for such expression constructs are known in the art and / or described herein. For example, expression vectors suitable for the methods of this disclosure in mammalian cells include, for example, vectors of pcDNA vector suites supplied by Invitrogen, vectors of pCI vector suites (Promega), pCMV vector suites (Clontech), pM vector (clone tech), pSI vector (promo), VP 16 vector (clone tech) or vector of pcDNA vector suite (Invitrogen).

Those skilled in the art will know, for example, a source of vectors such as Life Technologies Corporation, Clontech or Promega, and additional such vectors.

Methods for introducing isolated nucleic acids into cells for expression and gene structures containing them are known to those skilled in the art. The techniques used for a given organism depend on known successful techniques. Methods for introducing recombinant DNA into cells include, but are not limited to, microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes, such as lipofectamine (USA, Maryland, Zibko) PEG-mediated DNA inflow, electroporation, microparticle impact. Among others, DNA-coated tungsten or gold particles (Agra, Wisconsin, USA) Cetus).

Alternatively, the expression constructs of this disclosure are viral vectors. Suitable viral vectors are known in the art and are commercially available. Typical virus-based systems for delivery of nucleic acids and integration of nucleic acids into the host cell genome include, for example, retroviral vectors, lentiviral vectors or adeno-associated viral vectors. Alternatively, adenoviral vectors are useful for introducing nucleic acid that remains in the episome to the host cell. Viral vectors are an efficient and versatile method of gene delivery in target cells and tissues. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

For example, retroviral vectors generally contain a cis-acting long-chain repeat (LTR) with a packaging capacity of 6-10 kb or less of the foreign sequence. The minimal cis-acting LTR is then sufficient to replicate and package the vector, which is used to integrate the expression construct into the target cell to provide long-term expression. Widely used retroviral vectors include those based on murine murine leukemia virus (MuLV), gibbon monkey leukemia virus (GaLV), monkey immunodeficiency virus (SrV), human immunodeficiency virus (HIV), and combinations thereof For example, Sommerfelt et al., Virol 76: 58-59 (1992)), as described in [Buchscher et al. J. Virol. 56: 2731-2739 (1992); Johann et al. J. Virol 65: 1635-1640 (1990); Wilson et al. J. Virol 63: 274-2318 (1989); Miller et al. J. Virol 65: 2220-2224 (1991); PCT / US94 / 05700 Miller, Human Gene Therapy 7: 5-14, 1990; Scarpa et al., Virology 75: 849-852, 1991; Miller et al., BioTechniques 7: 980-990, 1989; And Burns et al., Proc. Natl Acad Sci USA 90: 8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have already developed for nucleic acid delivery. The AAV vector can be easily constructed using techniques known in the art. See, for example, U.S. Patent Nos. 5,173,414 and 5,139,941; International Publications WO 92/01070 and WO 93/03769; Lebkowski et al. Molec. Cell Biol 5: 3988-3996, 1988; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, Current Opinion in Biotechnology 5: 533-539, 1992; Muzyczka, Current Topics in Microbiol, and Immunol. 755: 97-129, 1992); Kotin, Human Gene Therapy 5: 793-801, 1994; See Shelling et al. Gene Therapy 7: 165-169, 1994); And Zhou et al. J Exp. Med. 779: 1867-1875, 1994).

Additional viral vectors useful for delivering the expression constructs of this disclosure include, for example, those derived from the pox family, such as vaccinia virus and avian poxvirus or alphavirus or conjugate viral vectors (see, for example, (Fisher-Hoch et al., Proc. Natl Acad Sci. USA 56: 317-321, 1989).

Foreigner Stem cell  Co-administration

Any of the peptides or stem cells used in the methods of the invention (e.g., stem cells expressing SDF-1 or protease-resistant mutant SDF-1 peptides) may be administered with foreign stem cells. Cells that can be administered with the peptides or genetically modified stem cells of the present invention include, but are not limited to, pluripotent or differentiating stem cells, or bone marrow cells. Examples of suitable exogenous stem cells include, but are not limited to, adult stem cells, mesenchymal progenitor cells (e.g., cells expressing the mesenchymal precursor cell marker STRO-1, for example, STRO-1 as described in US Publication No. 2014/0271567 ^Bright cells) and mesenchymal stem cells. In some embodiments, the exogenous stem cells may be homologous to the subject. In another embodiment, the exogenous stem cells may be autologous to the subject.

The exogenous stem cells may be mixed with the composition of the present invention immediately before or briefly before administration, or they may be co-cultured together for a certain period of time prior to administration. In other cases, exogenous stem cells may be administered separately from the peptides and / or stem cells of the present invention (e.g. stem cells expressing SDF-1 or protease-resistant SDF-1 peptides). The exogenous stem cells may be administered before or after the peptide or expressing stem cells.

In one embodiment, the composition to be administered to a subject can comprise an effective amount or a therapeutically or prophylactically effective amount of stem cells. An exemplary range of stem cells administered is from about 1x10 ³ cells / kg to about 1x10 ⁹ cells / kg (for example, 1x10 ³ cells / kg, 1x10 ⁴ cells / kg, 1x10 ⁵ cells / kg, 1x10 ⁶ cells / kg , 1 × 10 ⁷ cells / kg, 1 × ^{10 8} cells / kg, 1 × ^{10 9} cells / kg). For example, the composition may comprise about 1x10 ⁵ STRO-1 ⁺ cells / kg to about 1x10 ⁷ STRO-1 ⁺ cells / kg, or about 1x10 ⁶ to about 5x10 ⁶ STRO-1 ⁺ cells / kg. The precise amount of cells to be administered depends on various factors including the age, weight, sex of the patient, and the severity and degree of tissue damage in the subject.

In one embodiment, the cells are administered at a total number of cell doses regardless of the weight of the subject. For example, in some cases, stem cells may be in the range of about 50 million to 500 million cells (eg, 50 million, 100 million, 150 million, 200 million, 250 million, 250 million, 400 million, 450 million, or 500 million cells).

In some cases, the stem cells do not allow the cells to exit the circulation of the subject, but are contained within a chamber that allows the secreted by the cell to enter the circulation. In this manner, the solubility factor may be administered to the subject by allowing the cell to secrete the agent into the circulation of the subject. Such a chamber may be implanted at a site of the subject to increase the local level of the solubility factor, e.g., close to the site of tissue damage in the subject.

In some embodiments of the invention, immunosuppression of a subject prior to initiation of treatment with a composition comprising foreign stem cells may be necessary or undesirable. For example, transplantation of homologous, or even heterologous, STRO1 + cells or their offspring may be allowed in some cases.

In other embodiments, however, it may be desirable or appropriate to reduce the subject ' s immune response to a composition comprising a stem cell that is immunosuppressed and / or exogenous to the patient pharmacologically prior to initiation of cell therapy. This can be accomplished through the use of various, systemic or local immunosuppressive agents known in the art, or can be accomplished by delivering the cells in an encapsulated device, such as those described above. The cells can be encapsulated in capsules that are permeable to nutrients and oxygen that the cell needs, but which release the cells but are immune fluids and cell-impermeant therapeutic factor (s). For example, an encapsulant is hypoallergenic, easily and stably positioned in the target tissue, and provides added protection to the graft structure. These and other means of reducing or eliminating the immune response to the transplanted cells are known in the art. Alternatively, exogenous stem cells can be genetically modified to reduce their immunogenicity.

Pharmaceutical compositions and dosages

Any peptide or stem cell used in the methods of the present invention may be contained in any suitable amount in any suitable carrier material and the protease-resistant peptide or fusion protein will generally comprise from 1 to 95% , E. G., 5%, 10%, 20%, or 50%. The protease-resistant SDF-1 peptide or fusion protein described herein may be incorporated into a pharmaceutical composition comprising a carrier, such as, for example, saline, water, Ringer's solution and other drugs or excipients. The compositions are designed for intravenous delivery (e.g., by injection or implantable ports). Thus, the composition may be in the form of, for example, a suspension, emulsion, solution, or injection. All compositions can be prepared using standard methods in the art (see, for example, Remington's Pharmaceutical Sciences , 16th ed., A. Oslo. Ed., Easton, PA (1980)).

The peptides of the present invention may be delivered in a controlled-release or sustained-release system. For example, polymeric materials can be used to achieve controlled or sustained release of peptides (see, e. G., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NY (1984)]; U.S. Patents 5,679,377; 5,916,597; 5,912,015; 5,989,463 PCT Publication Nos. WO 99/15154 and WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co- (Polyvinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), polyacrylic acid Poly (lactide-co-glycolide) (PLGA), polyglutamic acid (PGA), and polyorthoesters.

It is anticipated that one skilled in the art will be able to adjust the dose of the peptide according to the case using methods that are well established in clinical medicine. Optimal dosages can be determined by methods known in the art and can be influenced by factors such as the age of the subject being treated, disease state, and other clinically relevant factors. In general, when administered to a human, the dosage of any of the therapeutic agents described herein (e.g., SDF-1 or protease-resistant mutant SDF-I peptides) will depend on the nature of the therapeutic agent and will be readily determined by those skilled in the art . Typically, such doses are usually about 0.001 to 2000 mg per day, preferably about 1 to 1000 mg per day, more preferably about 5 to 500 mg per day. In one embodiment, the dosage is from 0.01 mg / kg to 100 mg / kg, or preferably 1 mg / kg to 10 mg / kg per day (e.g., 1 mg / kg, 2 mg / / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg and 10 mg / kg).

The peptide or stem cell of the invention may be administered once, twice, three times, four times or five times daily; Once a week, twice a week, three times a week, four times a week, five times a week, or six times a week; Once a month, once every two months, once every three months, or once every six months; Or intravenously once a year. Alternatively, the peptides or stem cells of the present invention may be administered once or twice, and repeated administration may not be necessary. Administration of the peptides or stem cells described herein may continue until tissue damage (e. G., Tissue damage caused by myocardial infarction or peripheral vascular disease) is cured or ameliorated. The duration of treatment may be, for example, from one day to one week, from one week to one month, from one week to one year, or from one week to one year; Alternatively, the peptides or stem cells of the present invention may be administered for a shorter or longer duration. A continuous daily dose of the peptide or stem cells may not be necessary. The therapy may require a cycle in which the composition is not administered, or the treatment may be provided as needed.

The SDF-1 or mutant SDF-1 peptides or stem cells of the present invention may be administered at the exact time of tissue damage or after an initial onset of tissue damage or after a disease or condition (e.g., after myocardial infarction or acute organ damage, Acute kidney or liver injury) within minutes after initiation, recognition, or diagnosis. The SDF-1 or mutant SDF-1 peptides of the invention may be delivered after a short or long delay following initial tissue damage. For example, the SDF-1 or mutant SDF-1 peptides or stem cells of the invention can be administered within a few minutes to an hour, two hours, three hours , 4 hours, 6 hours, 12 hours, 24 hours, 48 hours or more, 3 days or more, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 1 month, 2 months, 3 months, 6 months , One year, or more than two years after the occurrence of the initial damage. Including, but not limited to, PVD, diabetic wounds, damage resulting from chronic organ damage (e.g., chronic kidney or liver injury) and inflammatory conditions (e.g., rheumatoid arthritis or Crohn's disease) For the tissue damage occurring over a period of time, the SDF-1 or mutant SDF-1 peptides or stem cells of the present invention can be tested immediately or after diagnosis of damage (e. G., PVD or diabetic wound) It can be delivered immediately after the sign. In this case, the delivery of the SDF-1 or mutant SDF-1 peptide or stem cells of the present invention may occur after a tissue injury has occurred or after the onset, recognition, or diagnosis of a tissue injury or disease or condition, , Weeks, three weeks, one month, two months, three months, four months, five months, six months, or even a year or more.

For any type of tissue injury, disease, or disorder described herein, the initial IV administration of the SDF-1 or mutant SDF-I peptides or stem cells of the present invention can be initiated by the initial onset of tissue damage, One year to two years after diagnosis or early detection of tissue damage, early onset of tissue damage, one to one year after diagnosis or diagnosis, early onset of tissue damage, one to six months after diagnosis or diagnosis , Early onset of tissue damage, one to six months after cognition or diagnosis, early onset of tissue damage, one to one month after cognition or diagnosis, early onset of tissue damage, one to one month after cognition or diagnosis, One week to two weeks after diagnosis or diagnosis, early onset of tissue damage, one hour to one week after recognition or diagnosis, early development of tissue damage, recognition or diagnosis Hour to three days, or an initial occurrence of tissue damage, from minutes to one hour after recognition or diagnosis.

The SDF-1 or mutant SDF-1 peptides or stem cells of the present invention may be delivered multiple times over the duration of the treatment, once or over the duration of the treatment. Depending on the severity of tissue damage, the SDF-1 or mutant SDF-1 peptides or stem cells of the invention may be delivered repeatedly over time to enable recovery or repair of damaged tissue.

In addition, intravenous delivery of SDF-1 or mutant SDF-1 peptides or stem cells of the present invention may be combined with additional forms of delivery of SDF-1 or mutant SDF-1 peptides or stem cells of the invention. In one embodiment, as after myocardial infarction, the SDF-1 or mutant SDF-I peptides or stem cells of the present invention may be initially delivered via an intracoronary or intraarterial route, followed by an intravenous route Followed by subsequent delivery of SDF-I or mutant SDF-I peptides or stem cells. In yet another embodiment, SDF-I or mutant SDF-I peptides or stem cells may be initially delivered via intramuscular or intramyocardial methods, followed by subsequent delivery of each treatment via an intravenous route . In any of these multiple delivery methods, intravenous administration may take place on days 1, 2, 3, 4, 5, 6, 7, 2, 1, 2, 3, 4 Months, 5 months, 6 months, 1 year or more. Here again, depending on the severity of the tissue damage, the SDF-1 or mutant SDF-1 peptides or stem cells of the present invention can be delivered repeatedly over time to enable repair or recovery of damaged tissue.

Appropriate doses of the peptides or stem cells used in the methods of the present invention depend on a number of factors, including the mode of administration, the severity of the disorder, the age, weight and health status of the subject being treated. Additionally, pharmacogenetic information (eg, pharmacokinetics, effects of genotypes on pharmacodynamics, or efficacy profile of treatment) on a particular subject can affect the dosage used.

Diagnosis and treatment

The methods of the present invention can be used for the treatment of any tissue damage (e. G., Tissue damage caused by damage to cardiac tissue or peripheral vascular disease resulting from myocardial infarction) or from any wound (e.g., diabetic wound) It is useful for treating the subject. Tissue damage may include, for example, cardiovascular conditions (e.g., myocardial infarction); Peripheral vascular disease (PVD); Peripheral arterial disease (PAD); Ulcers (e. G. Skin ulcers); Operation; Or diabetes. Tissue damage may also result from a CNS disorder or injury or inflammatory condition (e.g., rheumatoid arthritis, Crohn's disease, or graft versus host disease). The methods of the invention may also be used for the recovery or regeneration of organ damage (e.g., kidney or liver injury) resulting from disease or injury. The methods of the present invention can be used to promote wound healing or tissue repair. Those skilled in the art will appreciate that subjects of the present invention may have undergone standard testing and may have been identified at high risk for the presence of one or more risk factors without examination. Diagnosis of such disorders can be performed using any standard method known in the art.

The methods described herein are characterized by high concentrations of proteases (e.g., MMP-2, MMP-9, DPPIV, leukocyte elastase, cathepsin G, carboxypeptide degrading enzyme M and / or carboxypeptide degrading enzyme N) , Wherein the induction of stem cells upon administration of the protease-resistant SDF-1 peptide can induce regeneration or healing. Exemplary disorders to be treated with the compositions of the present invention include inflammatory and ischemic diseases (e. G., Myocardial infarction, stroke or lower limb ischemia), wound healing, and diabetic ulcers.

The efficacy of treatment may be determined by methods known to those skilled in the art, including, for example, symptom measurement of disease or disorder, physical examination, histopathological examination, blood chemistry analysis, computed tomography, cytological examination, magnetic resonance imaging ≪ / RTI > In certain embodiments, hemodynamic data is collected to measure the efficacy of the treatment. Hemodynamic tests may include, for example, measuring the ejection fraction (e.g., the fraction of blood pumping out of the ventricle in each heartbeat), measuring the expiratory term pressure, and the end-systolic volume (e. G. The volume of the blood). In one embodiment, hemodynamic testing can be used to monitor cardiac function in subjects suffering from tissue damage resulting from myocardial infarction or other forms of cardiac ischemia.

The methods of the present invention can be used in combination with additional treatments to promote wound healing or tissue repair. Therapeutic regimens that may be used in combination with the methods of the present invention include heparin, beta-blockers (e.g., atenolol, metoprolol, nadolol, oxprenolol, finsdolol, propranolol, or thymolol), angiotensin converting An angiotensin II receptor blocker (e. G., An angiotensin II receptor blocker), an angiotensin II receptor blocker (e. G., An angiotensin II receptor blocker) Aspirin, a cholesterol lowering agent (e.g., an HMG-CoA reductase inhibitor (e.g., a cholesterol lowering agent such as quercetin, fosartan, frovartan, ivesartan, losartan, olmesartan, telmisartan, or valsartan) Such as atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin), cell therapy, antiplatelet agents (e.g., clopidogrel, An antihypertensive agent, an antiarrhythmic agent (e.g., quinidine, procainamide, dyspyrimide, tricyclodecanil, tricyclodecanil, But are not limited to, lidocaine, mexiletine, tocainide, phenytoin, moricizine, flecainide, sotalol, ibutilide, amiodarone, bretylium, dofetilide, diltiazem or verapamil) Wound dressings, PDGF and / or sound pressure devices and treatments.

Example

The present invention is illustrated by the following examples, which are not intended to limit the present invention.

Example  1. Protease resistance SDF -One Mutant  Delayed and IV administration improves cardiac function in a rodent ischemia-reperfusion model

In the following examples, we have described an experiment demonstrating that intravenous delivery and prolonged delayed administration of mSDF-1 peptide-containing compositions improve cardiac function in an ischemic reperfusion model.

Rats were anesthetized with 0.05 mg / kg buprenorphine and 2-3% isoflurane. After intubation, between 4 and 5 ribs, the left anterior descending (LAD) coronary artery was ligated for 90 min. After 90 minutes, the suture was removed from the LAD to initiate reperfusion in the infarct zone. I closed the chest and skin of the mice afterwards. The mSDF-1 peptide was administered intravenously after 7 days of infarction (> 15 mice per group). For intravenous injection, 100 μl of S-SDF-1 (S4V) in PBS (at doses of 0, 0.1 and 1.0 mg / kg) was injected into the tail vein of the mice.

In each of the above experiments, the hemodynamic function of the rats was analyzed by randomized and blind study 4 weeks after intravenous administration (5 weeks after ischemia reperfusion injury). Rats were anesthetized with 0.05 mg / kg buprenorphine and 2-3% isoflurane. A 16G tube was inserted into the rats and mechanical ventilation was started. The left carotid cannula was cannulated with PE 10 to deliver hyperosmolar saline (50 μl of 25% NaCl solution in water). High osmolarity saline was used to measure the parallel conductivity of the volumetric measurements.

To measure ejection fraction (EF) and intra-abdominal pressure, the right carotid artery was cannulated. A pressure-volume catheter was inserted and allowed to pass through the left ventricle. Baseline pressure-volume measurements were obtained. High osmolar saline (above) was injected into the carotid artery, followed by pressure-volume measurements.

Our results show that intravenous injection of S-SDF-1 (S4V) after 7 days of ischemia reperfusion injury resulted in a 10% reduction in rat mortality compared to PBS control (Fig. 1).

Example  2. Protease resistance SDF -One Mutant  Delayed and IV Administration  Ischemia Reperfusion  Heart function in the model Improve

We also measured the effect of intravenous delivery and delayed administration of mSDF-1 peptide-containing compositions on cardiac function in a micro-Yucatan swine infarction model.

In these experiments, the pigs were anesthetized and their left anterior descending (LAD) coronary arteries blocked with a balloon catheter. After 90 minutes, the balloon catheter was removed from the LAD to initiate reperfusion in the infarct area. I closed the chest and skin of the pig. Randomized and blinded studies were performed immediately after ischemia with a PBS control or mSDF-1 peptide (at 1 mg / kg or 3 mg / kg) via intra-coronary administration (n = 5 pigs). At 4 weeks (one month) after infarction, a second dose of PBS control or mSDF-1 peptide (at 1 mg / kg or 3 mg / kg) was administered intravenously.

In each of the above experiments, the ejection fraction (EF) was measured at 4 weeks after infarction, 8 weeks after infarction, and 12 weeks after infarction. Our results demonstrated a significant improvement in EF at 12 weeks after pig infarction with 3 mg / kg of mSDF-1 peptide (Fig. 2). Specifically, we observed a 2.7% absolute EF improvement at 12 weeks after pig infarction with 3 mg / kg mSDF-1 peptide compared to the control (1-sided T-test, p & Respectively. The lower dose group (1 mg / kg mSDF-1 peptide) showed a clear trend towards EF improvement, lasting 4-12 weeks (Figure 2).

Other embodiments

It is clear from the foregoing description that changes and modifications may be made to the invention described herein for various applications and conditions. Such embodiments are also within the scope of the following claims.

All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference to the same extent as if each respective independent publication or patent application was specifically and individually indicated to be incorporated by reference.

                         SEQUENCE LISTING &Lt; 110 > Mesoblast International S et al.   <120> METHODS FOR REPAIRING TISSUE DAMAGE USING PROTEASE-RESISTANT        MUTANTS OF STROMAL CELL DERIVED FACTOR-1 <130> 50591-009WO2 <140> PCT / US2014 / 070010 <141> 2014-12-12 &Lt; 150 > US 61 / 915,842 <151> 2013-12-13 <160> 77 <170> PatentIn version 3.5 <210> 1 <400> 1 000 <210> 2 <400> 2 000 <210> 3 <400> 3 000 <210> 4 <400> 4 000 <210> 5 <400> 5 000 <210> 6 <400> 6 000 <210> 7 <400> 7 000 <210> 8 <400> 8 000 <210> 9 <400> 9 000 <210> 10 <400> 10 000 <210> 11 <400> 11 000 <210> 12 <400> 12 000 <210> 13 <400> 13 000 <210> 14 <400> 14 000 <210> 15 <400> 15 000 <210> 16 <400> 16 000 <210> 17 <400> 17 000 <210> 18 <400> 18 000 <210> 19 <400> 19 000 <210> 20 <400> 20 000 <210> 21 <400> 21 000 <210> 22 <400> 22 000 <210> 23 <400> 23 000 <210> 24 <400> 24 000 <210> 25 <400> 25 000 <210> 26 <400> 26 000 <210> 27 <400> 27 000 <210> 28 <400> 28 000 <210> 29 <400> 29 000 <210> 30 <400> 30 000 <210> 31 <400> 31 000 <210> 32 <400> 32 000 <210> 33 <400> 33 000 <210> 34 <400> 34 000 <210> 35 <400> 35 000 <210> 36 <400> 36 000 <210> 37 <400> 37 000 <210> 38 <400> 38 000 <210> 39 <400> 39 000 <210> 40 <400> 40 000 <210> 41 <400> 41 000 <210> 42 <400> 42 000 <210> 43 <400> 43 000 <210> 44 <400> 44 000 <210> 45 <400> 45 000 <210> 46 <400> 46 000 <210> 47 <400> 47 000 <210> 48 <400> 48 000 <210> 49 <400> 49 000 <210> 50 <400> 50 000 <210> 51 <400> 51 000 <210> 52 <211> 68 <212> PRT <213> Homo sapiens <400> 52 Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 53 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> MOD_RES &Lt; 222 > (3) <223> Xaa is any amino acid <400> 53 Lys Pro Xaa Xaa Xaa Xaa Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 54 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 54 Lys Pro His Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 55 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 55 Lys Pro Cys Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 56 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 56 Lys Pro Val Ser Thr Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 57 <400> 57 000 <210> 58 <400> 58 000 <210> 59 <400> 59 000 <210> 60 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 60 Lys Pro Val Ser Val Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 61 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 61 Lys Pro Val Ser Leu Cys Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 62 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 62 Lys Pro Val Ser Leu Gly Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 63 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 63 Lys Pro Val Val Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 64 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 64 Lys Pro Val Ser Ser Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 65 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 65 Lys Pro Val Val Ser Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro             20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln         35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys     50 55 60 Ala Leu Asn Lys 65 <210> 66 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 66 Gly Gly Gly Gly Ser 1 5 <210> 67 <211> 302 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 67 Ser Lys Pro Val Val Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys Gly Gly Gly Gly Ser Val Asp Lys Thr His Thr 65 70 75 80 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe                 85 90 95 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             100 105 110 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         115 120 125 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     130 135 140 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 145 150 155 160 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 165 170 175 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser             180 185 190 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         195 200 205 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val     210 215 220 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 225 230 235 240 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 245 250 255 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             260 265 270 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         275 280 285 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     290 295 300 <210> 68 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> MOD_RES <222> (4) (7) <223> Xaa is any amino acid <400> 68 Ser Lys Pro Xaa Xaa Xaa Xaa Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 69 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 69 Ser Lys Pro Val Val Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 70 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 70 Ser Lys Pro Val Ser Ser Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 71 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 71 Ser Lys Pro Val Val Ser Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 72 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 72 Ser Lys Pro His Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 73 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 73 Ser Lys Pro Cys Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 74 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 74 Ser Lys Pro Val Ser Thr Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 75 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 75 Ser Lys Pro Val Ser Ser Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 76 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 76 Ser Lys Pro Val Ser Leu Cys Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65 <210> 77 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 77 Ser Lys Pro Val Ser Leu Gly Tyr Arg Cys Pro Cys Arg Phe Phe Glu 1 5 10 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr             20 25 30 Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg         35 40 45 Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu     50 55 60 Lys Ala Leu Asn Lys 65

Claims (40)

Mutation SDF-1 (mSDF-1) , stromal cell-derived factor-1 comprising the formula _{mSDF-1-Y z, X} p -mSDF-1, or _{X p -mSDF-1-Y z} (SDF-1) Comprising administering intravenously a composition comprising an isolated mutant form of a peptide or a stem cell expressing said composition,
Wherein said SDF-1 is a peptide comprising the amino acid sequence of at least amino acids 1-8 of SEQ ID NO: 53 and optionally extends at the C-terminus by all or any portion of the remaining sequence of SEQ ID NO: 53,
SEQ ID NO: 53 has the amino acid sequence:
KPX ₃ X ₄ X ₅ X ₆ YRCPCRFFESHVARANVKHLK ILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALNK (SEQ ID NO: 53) wherein X ₃ , X ₄ , X ₅ and X ₆ are any amino acid,
a) X _p is a proteinaceous amino acid (s) or a protease protecting organic group, p is any integer of 1 to 4,
b) Y _z is a proteinaceous amino acid (s) or a protease protecting organic group, z is any integer from 1 to 4,
c) said mSDF-1 or said mSDF-1-Y _z retains the chemoattractant activity against T cells and inhibits matrix metalloproteinase-2 MMP-2), matrix metalloproteinase-9 (MMP-9), leukocyte elastase and / or cathepsin G,
d) said X _p -mSDF-1 or said X _p -mSDF-1-Y _z retaining chemoattractant activity against T cells and at a rate that is at least 50% less than the rate at which native SDF- MMP-9, leukocyte elastase, and / or cathepsin G at a rate that is inactivated by Peptide Enzymol IV (DPPIV) and is at least 50% less than the inactivity rate of native SDF-1. Activated,
Wherein the isolated mutant form of SDF-1 is administered intravenously in an amount sufficient to treat or ameliorate tissue damage in a subject in need thereof.
In the subject, a method of treating or ameliorating tissue damage resulting from a disease or condition. The method of claim 1, wherein the mutated form of the SDF-1 peptide does not comprise an amino acid sequence of at least amino acids 1-8 of SEQ ID NO: 52. 3. A method according to claim 1 or 2, wherein X ₃ is valine, histidine, or cysteine manner. 3. The method according to claim 1 or 2, wherein X &lt; ₄ &gt; is serine or valine. 5. The method according to any one of claims 1 to 4, wherein X &lt; ₅ &gt; is leucine, proline, threonine, or valine. Any one of claims 1 to A method according to any one of items 5, X ₆ is a method of serine, cysteine, or glycine. 7. The method according to any one of claims 1 to 6, wherein the peptide is an X _p -mSDF-1 peptide or an X _p -mSDF-1-Y _z peptide wherein X is serine and p is 1. 7. The method according to any one of claims 1 to 6, wherein the peptide is an mSDF-1-Y _z peptide or an X _p -mSDF-1-Y _z peptide wherein Y is serine and z is 1. 9. The method according to any one of claims 1 to 8, wherein said mutant form of SDF-1 is a mutated form of SDF-1, wherein n is an integer from 0 to 3, and L is a 3-9 amino acid how the linker sequence and, an Fc fusion protein comprising the Fc peptide of the formula A- (L) _n -Fc from the Fc region of an immunoglobulin. 10. The method of claim 9, wherein n is 1 and L is GGGGS (SEQ ID NO: 66). 11. The method according to any one of claims 1 to 10, wherein the stem cells expressing the mutant form of SDF-1 are mesenchymal stem cells or mesenchymal precursor cells. 12. The method according to any one of claims 1 to 11, further comprising the step of administering foreign stem cells. 13. The method according to claim 12, wherein the foreign stem cells are mesenchymal stem cells or mesenchymal precursor cells. 14. The method of any one of claims 1 to 13 wherein said disease or condition is selected from the group consisting of stroke, lower ischemia, tissue damage due to trauma, myocardial infarction, peripheral vascular disease, chronic heart failure, diabetes, diabetic wound healing, Disease or injury, CNS disease or injury and inflammatory condition. 15. The method of claim 14, wherein the disease or condition is myocardial infarction. 15. The method of claim 14, wherein said disease or condition is peripheral vascular disease. 15. The method of claim 14, wherein said disease or condition is diabetes. 15. The method of claim 14, wherein the disease or condition is diabetic wound healing. 15. The method of claim 14, wherein the organ disease or injury is kidney or liver disease or injury. 15. The method of claim 14, wherein the inflammatory condition is rheumatoid arthritis, Crohn's disease, or graft versus host disease. 21. The method according to any one of claims 1 to 20, wherein said stem cell or composition is administered to a peripheral or central vein. 22. The method according to any one of claims 1 to 21, wherein said stem cell or composition is administered within minutes after the onset of said disease, condition, or tissue damage. 22. The method according to any one of claims 1 to 21, wherein said stem cell or composition is administered within 12 hours after the onset of said disease, condition, or tissue damage. 22. The method according to any one of claims 1 to 21, wherein said stem cell or composition is administered at least 24 hours or more after the onset or diagnosis of said disease, condition, or tissue damage. 22. The method according to any one of claims 1 to 21, wherein said stem cell or composition is administered at least 48 hours after the onset or diagnosis of said disease, condition, or tissue damage. 22. The method according to any one of claims 1 to 21, wherein said stem cell or composition is administered at least 7 days after the onset or diagnosis of said disease, condition, or tissue damage. 22. The method according to any one of claims 1 to 21, wherein the stem cell or composition is administered at least one month after the onset or diagnosis of the disease, condition, or tissue damage. 22. The method according to any one of claims 1 to 21, wherein the stem cell or composition is administered more than six months after the onset or diagnosis of the disease, condition, or tissue damage. 29. The method according to any one of claims 1 to 28, wherein said method is used in combination with intra-arterial administration of stem cells expressing SDF-1 or a mutant SDF-1 peptide, or SDF-1 or a mutant SDF-1 peptide . 30. The method of claim 29, wherein the intra-arterial administration occurs prior to intravenous administration. 31. The method according to any one of claims 22 to 30, wherein the disease or condition is tissue damage due to trauma, organ disease, inflammatory disease, myocardial infarction, or peripheral vascular disease. 31. The method according to any one of claims 22 to 30, wherein said disease or condition is cardiovascular disease. 33. The method according to any one of claims 1 to 32, wherein said stem cell or composition is administered more than once until said tissue damage is reduced, restored or new blood vessel formation occurs. 33. The method of any one of claims 1 to 32, wherein the stem cell or composition is administered more than once to improve one or more symptoms of the disease or condition. 35. The method of any one of claims 1 to 34, wherein the tissue is cardiac tissue. 35. The method of any one of claims 1 to 34, wherein the tissue is vascular tissue. 35. The method according to any one of claims 1 to 34, wherein the tissue is an organ tissue. 38. The method of claim 37, wherein the organ is the kidney or liver. 39. The method according to any one of claims 1 to 38, wherein the mutated form of SDF-1 comprises the sequence of SEQ ID NO: 67. 39. The method of any one of claims 1 to 38, wherein the SDF-1 comprises the sequence of SEQ ID NO: 69.

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